Fusion protein and Enterokinase

Ted Michelini tedm at darkwing.uoregon.edu
Sun Apr 27 01:19:47 EST 1997

In article < at chemvx.tamu.edu>,
khuang at CHEMVX.CHEM.TAMU.EDU (Huang Ke-xue) wrote:

> Hi, Netters,
> I am experessing some genes in E. coli by using pET32, it gave me good
> result. But the painful problem is when I tried to relase the native protein
> from fusion protein using enterokinase, I could not get the amount what I
> want although I followed the protocol Novagen provided. Sometimes I found
> the protein were degraded (by SDS-PAGE).
> Is it a usual problem for fusion protein or my hands are two bad? 
> Thanks!
> Huang 
Are you using the cloned enterokinase?
There may be other problems Huang, but check the activity of the Novagen
single chain, Pichia expresssed enterokinase, it is far less active than
the mammalian derived enzyme available from a number of other sources.
Specific proteases are sometimes real dogs; they are barely catalytic. The
worst case being maybe factor X, then enterokinase, followed by thrombin.
Try some of the purified native enterokinase and see if it works better.
good luck

Ted Michelini
Institute of Molecular Biology
University of Oregon

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